This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The goal of this project is to fully explore the physiological functions of autophagy. For this, it is critical to understand the central part of the autophagy pathway, the autophagosome formation process. To date, approximately 30 autophagy-related (ATG) genes have been identified using yeast model systems. Among them, 15 are conserved from yeast to human and are essential for autophagosome formation;the products of these genes are termed the core machinery proteins. It was hypothesized that these core machinery proteins function at the phagophore assembly site (PAS). The PAS is essentially uncharacterized, even though it is thought to play a fundamental role in autophagosome biogenesis. Thus, there are basic questions such as the membrane nature of this site, whether the phagophore expands by vesicular fusion, and whether the Atg proteins (almost all of which associate in large part with the PAS) are in complexes at particular locations at the PAS, such as at the growing ends of the phagophore (note that the exact relationship between the PAS and the phagophore is not known;the PAS may develop into a phagophore, or may provide membrane to a separate site that corresponds to the phagophore).